Antibacterial peptide, aliphatic chain modified antibacterial peptide, synthetic method and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to an antibacterial peptide, an aliphatic chain modified antibacterial peptide, a synthesis method and application. The amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO. 1. The amino acid sequence of the aliphatic chain modified antibacterial peptide is as shown in SEQ ID NO. 2. The antibacterial peptide and the aliphatic chain modified antibacterial peptide can efficiently kill multi-drug-resistant bacteria, have good stability, are not easy to cause drug resistance, are low in hemolytic toxicity, and have wider clinical application potential.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an antimicrobial peptide, a fat chain-modified antimicrobial peptide, a synthesis method and uses thereof. Background Art

[0002] Antimicrobial peptides (also known as host defense peptides) are important barriers for hosts to resist the invasion of pathogenic microorganisms and are widely present in microorganisms, plants and animals. They usually have moderate direct antimicrobial activity under physiological conditions and are composed of rich and diverse amino acid sequences. Among them, the content of cationic residues such as arginine and lysine is high, which makes antimicrobial peptides carry positive charges in a neutral pH environment; at the same time, these peptides often contain 50% or more hydrophobic amino acids. The positive charge and high hydrophobicity together promote the folding of antimicrobial peptides to form an amphipathic α-helical structure: hydrophobic residues are located on one side of the molecule, while cationic and polar residues are concentrated on the other side. Given their key role in the innate immune system, antimicrobial peptides have been regarded as potential new antibiotic candidate molecules in the past two decades. However, existing antimicrobial peptides still face some technical bottlenecks in clinical applications. First, many antimicrobial peptides, despite their strong antimicrobial activity during long-term use, are limited in their clinical safety due to their easy cytotoxicity. Secondly, although antimicrobial peptides can effectively kill a variety of bacteria, there are still certain problems in terms of stability and hemolytic toxicity. Some antimicrobial peptides have poor stability and are difficult to maintain long-lasting efficacy. At the same time, some antimicrobial peptides may lead to the development of drug resistance, reducing their therapeutic effects.

[0003] Therefore, how to design an antimicrobial peptide with broad-spectrum antibacterial properties, which can effectively kill clinical multidrug-resistant bacteria and has low hemolytic toxicity and cytotoxicity, has become an important technical problem that needs to be urgently solved in the current research on antimicrobial peptides. Summary of the invention

[0004] Based on the above technical problems, the present invention introduces amino acids such as arginine, lysine, valine and leucine in the sequence design to synthesize a highly efficient broad-spectrum antimicrobial peptide, which can effectively kill multidrug-resistant bacteria, has good stability, is not easy to induce drug resistance, and has low hemolytic toxicity, and has broader clinical application potential.

[0005] The specific technical solutions provided by the present invention are as follows:

[0006] In a first aspect, the present invention provides an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The second aspect of the present invention provides a method for synthesizing the antimicrobial peptide, comprising the following steps:

[0008] After removing the Fmoc protecting group from the α-helical peptide chain in conotoxin GeXIVA, amino acids are successively coupled in the order shown in SEQ ID NO.1 and iterated until the extension of the target peptide chain is completed, and then the protecting groups are removed to obtain the antibacterial peptide.

[0009] As a preferred embodiment of the present invention, 5-fold equivalent of amino acid is used in each step when successively coupling amino acids, and the reaction time is 5-10 minutes.

[0010] In the third aspect of the present invention, there is provided a fatty acid chain-modified antibacterial peptide, which is obtained by coupling a fatty acid with the exposed amino group of the antibacterial peptide, and its amino acid sequence is as shown in SEQ ID NO.2.

[0011] As a preferred embodiment of the present invention, after the coupling reaction is completed, the coupling reaction product is separated by the ether precipitation method to obtain a crude peptide.

[0012] As a preferred embodiment of the present invention, the synthesis of the fatty acid chain-modified antibacterial peptide further includes purification. The specific operation steps are as follows: trifluoroacetic acid and water are mixed at a volume ratio of 0.01:100 as mobile phase A, and acetonitrile and water are mixed at a volume ratio of 3:2 as mobile phase B, and gradient elution separation is carried out by high performance liquid chromatography. During the gradient elution process, mobile phase B linearly changes from 5% to 70% in 0-65 minutes; trifluoroacetic acid and water are mixed at a volume ratio of 0.1:100 as mobile phase A, and acetonitrile and water are mixed at a volume ratio of 9:1 as mobile phase B, and the purity of the separated compound is detected by reverse phase chromatography. Mobile phase B linearly changes from 5% to 70% in 0-30 minutes; the compound with a purity >95% is collected and dried to obtain the fatty acid chain-modified antibacterial peptide.

[0013] In the fourth aspect of the present invention, there is provided the use of the antibacterial peptide or the fatty acid chain-modified antibacterial peptide in the preparation of an antibacterial product.

[0014] As a preferred embodiment of the present invention, the antibacterial effect is against Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa.

[0015] More preferably, the antibacterial effect is against drug-resistant Staphylococcus aureus.

[0016] In the fifth aspect of the present invention, there is provided an antibacterial product, which uses the antibacterial peptide or the fatty acid chain-modified antibacterial peptide as the only active ingredient.

[0017] As a preferred embodiment of the present invention, the antibacterial product is formulated by compounding the antibacterial peptide or the fatty acid chain-modified antibacterial peptide and a pharmaceutically acceptable excipient.

[0018] As a preferred embodiment of the present invention, the antibacterial product uses the combination of the antimicrobial peptide and the fatty chain-modified antimicrobial peptide as the sole active ingredient.

[0019] More preferably, the antibacterial product is prepared by compounding the antimicrobial peptide, the fatty chain-modified antimicrobial peptide, and a pharmaceutically acceptable excipient.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1. In the sequence design of the present invention, amino acids such as arginine, lysine, valine, and leucine are introduced to synthesize the antimicrobial peptide. As positively charged amino acids, arginine and lysine enhance the binding force between the polypeptide and the cell membrane through electrostatic interaction; as hydrophobic amino acids, valine and leucine improve the overall hydrophobicity of the polypeptide, thereby further enhancing its affinity and penetration ability for the cell membrane and enhancing the antibacterial effect.

[0022] 2. Since the antimicrobial polypeptide is rich in cationic residues such as arginine and lysine, it is more likely to be recognized and degraded by specific proteases in vivo, resulting in a shortened half-life and reduced activity. To address this problem, the present invention further modifies the N-terminus of the antimicrobial peptide with a fatty chain, thereby enhancing the stability of the polypeptide in the physiological environment and expecting to maintain its antibacterial activity to the greatest extent, thus obtaining the fatty chain-modified antimicrobial peptide with the best comprehensive performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 are the liquid chromatography and mass spectrometry diagrams of the antimicrobial peptide synthesis. (A) Liquid chromatography diagram of VG7; (B) Mass spectrometry diagram of VG7; (C) Liquid chromatography diagram of C4-VG7; (D) Mass spectrometry diagram of C4-VG7.

[0024] Figure 2 is the stability test of the antimicrobial peptide; (A) Simulated gastric juice; (B) 80°C; (C) pH.

[0025] Figure 3 is the MIC determination of the antimicrobial peptide; (A) MIC determination of the antimicrobial peptide and common antibiotics; (B) MIC determination of the antimicrobial peptide against clinical multi-drug resistant Acinetobacter baumannii;

[0026] Figure 4 is the time-kill curve of the antimicrobial peptides VG7 and C4-VG7 against four bacteria; (A) MRSA; (B) Acinetobacter baumannii; (C) Klebsiella pneumoniae; (D) Pseudomonas aeruginosa.

[0027] Figure 5VG7 and C4-VG7 inhibited bacterial biofilm formation in a concentration-dependent manner; (A-D) were the inhibitions of antimicrobial peptide VG7 on the biofilm formation of MRSA, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa, respectively; (E-H) were the inhibitions of antimicrobial peptide C4-VG7 on the biofilm formation of MRSA, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa, respectively.

[0028] Figure 6 The effects of antimicrobial peptides VG7 and C4-VG7 on the eradication of biofilms formed by four bacteria; (A-D) were the eradications of antimicrobial peptide VG7 on the formed biofilms of MRSA, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa, respectively; (E-H) were the eradications of antimicrobial peptide C4-VG7 on the formed biofilms of MRSA, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa, respectively.

[0029] Figure 7 The evaluation of the drug resistance of antimicrobial peptides VG7 and C4-VG7 against four bacteria; (A) the changes in MIC of different drugs against MRSA; (B) the changes in MIC of different drugs against Acinetobacter baumannii; (C) the changes in MIC of different drugs against Klebsiella pneumoniae; (D) the changes in MIC of different drugs against Pseudomonas aeruginosa.

[0030] Figure 8 Hemolytic toxicity assay.

[0031] Figure 9 Observation of the effects of antimicrobial peptides VG7 and C4-VG7 on the morphology of four bacteria by scanning electron microscopy; (A) MRSA; (B) Acinetobacter baumannii; (C) Klebsiella pneumoniae; (D) Pseudomonas aeruginosa.

[0032] Figure 10 The effects of antimicrobial peptide C4-VG7 on the outer membrane permeability of three bacteria; (A) Acinetobacter baumannii; (B) Klebsiella pneumoniae; (C) Pseudomonas aeruginosa.

[0033] Figure 11 The effects of antimicrobial peptide C4-VG7 on the inner membrane permeability of four bacteria; (A) MRSA; (B) Acinetobacter baumannii; (C) Klebsiella pneumoniae; (D) Pseudomonas aeruginosa.

[0034] Figure 12 The effects of antimicrobial peptide C4-VG7 on the membrane potential of four bacteria; (A) MRSA; (B) Acinetobacter baumannii; (C) Klebsiella pneumoniae; (D) Pseudomonas aeruginosa.

[0035] Figure 13It is the binding situation of antibacterial peptide C4-VG7 with four kinds of bacterial DNAs; (A) MRSA; (B) Acinetobacter baumannii; (C) Klebsiella pneumoniae; (D) Pseudomonas aeruginosa.

[0036] Figure 14 It is the efficacy of the antibacterial peptide on the acute peritoneal inflammation model induced by Acinetobacter baumannii; (A) Body weight change; (B) Survival rate; (C) Liver colony count; (D) Spleen colony count; (E) Lung colony count; (F) Kidney colony count.

[0037] Figure 15 It is the efficacy of the antibacterial peptide on the mouse model of skin infection by Acinetobacter baumannii; (A) Body weight change; (B) Skin colony count.

[0038] Figure 16 It is the in vivo safety evaluation of the antibacterial peptide; (A) Body weight change; (B) AST content; (C) ALT content; (D) ALB content; (E) BUN content; (F) CREA content; (G) UA content; (H) HE staining of visceral sections. Detailed implementation manners

[0039] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the drawings, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The reagents or instruments used in the present invention, if not specified for the manufacturer, are all conventional products that can be obtained through commercial purchase.

[0040] Antibacterial polypeptides are important barriers for hosts to resist the invasion of pathogenic microorganisms and are widely present in microorganisms, plants and animals. They usually have moderate direct antibacterial activity under physiological conditions and are composed of rich and diverse amino acid sequences. Among them, the content of cationic residues such as arginine and lysine is relatively high, making the antibacterial polypeptides carry a positive charge in a neutral pH environment; at the same time, these polypeptides often contain 50% or more hydrophobic amino acids. The positive charge and high hydrophobicity together promote the antibacterial polypeptides to fold into an amphiphilic α-helical structure: the hydrophobic residues are located on one side of the molecule, while the cationic and polar residues are aggregated on the other side. Given their key role in the innate immune system, antibacterial polypeptides have been regarded as potential new antibiotic candidate molecules in the past two decades.

[0041] In the previous research of the invention team, it was found that the α-helix structure (Cys9-Cys20) in conotoxin GeXIVA and the presence of a large number of arginines in the sequence endow it with high potential antibacterial activity. Based on this discovery, amino acids such as arginine, lysine, valine, and leucine were introduced in the sequence design. As positively charged amino acids, arginine and lysine enhance the binding force between the polypeptide and the cell membrane through electrostatic interaction; as hydrophobic amino acids, valine and leucine improve the overall hydrophobicity of the polypeptide, thereby further enhancing its affinity for and penetration ability into the cell membrane and strengthening the antibacterial effect.

[0042] However, since antimicrobial polypeptides are rich in cationic residues such as arginine and lysine, they are more likely to be recognized and degraded by specific proteases in vivo, resulting in a shortened half-life and reduced activity. Based on this, the N-terminus of the antimicrobial peptide VG7 was modified with a fatty chain in the present invention to enhance the stability of the polypeptide in the physiological environment and expect to maintain its antibacterial activity to the greatest extent, and thus the antimicrobial peptide C4-VG7 was obtained.

[0043] Example 1

[0044] Synthesis of antimicrobial peptides and fatty chain-modified antimicrobial peptides

[0045] 1. Synthesis

[0046] In the present invention, a LibertyBlue microwave-assisted polypeptide synthesizer based on the Fmoc-SPPS method was used to synthesize the crude antimicrobial peptide. The resin used was Rink amide-MBHA-Resin, and all amino acids were protected with standard Fmoc protecting groups. The synthesis process is as follows:

[0047] (1) The C-terminus of the α-helix structure (i.e., Cys9-Cys20) in conotoxin GeXIVA was fixed on the Rink amide-MBHA resin carrier, fully swollen in N,N-dimethylformamide (DMF), and then the Fmoc protecting group was removed by deprotection using a 20% (v / v) piperidine / DMF solution.

[0048] (2) Activate the amino acid using ethyl 2-cyano-2-oxoacetate / N,N'-diisopropylcarbodiimide (Oxyma / DIC), couple the activated amino acid with the deprotected resin peptide to form an amino acid ligation cycle, and repeat the above steps in the order from the C-terminus to the N-terminus of the amino acid sequence (VKRLLKRLRKLV) (i.e., valine, leucine, lysine, arginine, leucine, arginine, lysine, leucine, leucine, arginine, leucine, valine) until the coupling of the last amino acid is completed, and then use a 20% (v / v) piperidine / DMF solution for deprotection. This iterative process continues until the extension of the target peptide chain is completed, and finally, the side-chain protecting groups are removed and the resin is dissociated simultaneously through a cleavage system of TFA / TIS / Water / DODT (92.5 / 2.5 / 2.5 / 2.5, v / v / v / v).

[0049] The cleavage reaction was carried out in a CEM Razor high-throughput system (40 °C, 30 minutes).

[0050] The post-treatment of the product was carried out by gradient ice-ether precipitation method, specifically: inject the cleavage solution into 40 mL of ice-ether and let it stand at 4 °C for 2 hours, then centrifuge 3 times at 12000 rpm and 4 °C for 15 minutes each time, and finally obtain a pale yellow linear peptide powder, that is, the crude antibacterial peptide, after drying.

[0051] The synthesis of the fatty acid chain-modified antibacterial peptide was carried out after the completion of the main chain synthesis, and the specific steps were as follows:

[0052] After the coupling of the last amino acid was completed, a 20% (v / v) piperidine / DMF solution was used for deprotection to remove the Fmoc protecting group. The coupling reaction of butyric anhydride with the exposed amino group was mediated by a HATU / DIPEA catalytic system. The reaction used DMF as the solvent, and the reaction was shaken for 1.5 hours, and then the side-chain protecting groups were removed and the resin was dissociated simultaneously through a cleavage system of TFA / TIS / Water / DODT (92.5 / 2.5 / 2.5 / 2.5, v / v / v / v), and finally the crude fatty acid chain-modified antibacterial peptide was obtained by gradient ice-ether precipitation method.

[0053] The crude antibacterial peptides and the crude fatty acid chain-modified antibacterial peptides obtained above were purified respectively according to the following process:

[0054] Dissolve them separately by adding 5 mL of mobile phase B (60% (v / v) acetonitrile / H2O), dilute with 100 mL of mobile phase A (0.1% (v / v) trifluoroacetic acid / H2O), filter through a 0.45 μm filter membrane, and then purify by preparative RP-HPLC. Preparative RP-HPLC used an XBridge Peptide BEH C18 chromatographic column ( 5 μm, 19 × 100 mm), with mobile phase B having a linear gradient change from 5% to 70% within 65 minutes at a flow rate of 12 mL / min. The collected fractions were analyzed by a Waters TQD ultra performance liquid chromatography - mass spectrometry instrument. On a Waters e2569 high performance liquid chromatography instrument, the separated compounds were monitored for purity using a reversed - phase C18 chromatographic column Symmetry Shield RP18 column( 5 μm, 4.6 × 150 mm) at a flow rate of 1 mL / min at 214 nm. The mobile phase B had a gradient change from 5% to 70% within 30 minutes (mobile phase A: 0.1% trifluoroacetic acid / H2O, v / v; mobile phase B: 90% acetonitrile / H2O, v / v)). Compounds with a purity > 95% were collected, freeze - dried, weighed, and the obtained antimicrobial peptide pure product was labeled as VG7, and the fatty - chain - modified antimicrobial peptide was labeled as C4 - VG7. The yield was calculated and used for subsequent bioactivity identification.

[0055] The amino acid sequence of antimicrobial peptide VG7 is shown in SEQ ID NO.1: VKRLLKRLRKLV # , # representing the C - terminal amidation of the polypeptide; the structural formula is as follows:

[0056]

[0057] The amino acid sequence of the fatty - chain - modified antimicrobial peptide C4 - VG7 is shown in SEQ ID NO.2:

[0058] NVKRLLKRLRKLV # , N represents C3H7CO, # representing the C - terminal amidation of the polypeptide; the structural formula is as follows:

[0059]

[0060] 2. Physicochemical properties

[0061] See Table 1.

[0062] Table 1 Physicochemical properties of antimicrobial peptides

[0063]

[0064] 3. Liquid chromatography and mass spectrometry diagrams of antimicrobial peptide synthesis

[0065] The liquid chromatography data and mass spectrometry data are shown in Figure 1 .

[0066] From Figure 1It can be seen that the purity of both antibacterial peptides VG7 and C4-VG7 is greater than 95%, and the molecular weight identified by mass spectrometry is consistent with the theoretical molecular weight.

[0067] 4. Stability of Antibacterial Peptides

[0068] 4.1 Stability in Gastric Juice

[0069] The simulated gastric juice was prepared according to the United States Pharmacopeia (USP 35-NF 30 (2012) 1174-1666 Dietary Supplements). The specific method is as follows:

[0070] Take 2.0 g of sodium chloride and 3.2 g of pepsin (labeled with 800 - 2500 activity units per mg), add 7.0 mL of hydrochloric acid and water to dissolve and make up to 1000 mL. The pH value of this solution should be about 1.2.

[0071] Dissolve antibacterial peptides VG7 and C4-VG7 in the prepared solution respectively to make the final concentration 0.25 mM, incubate at 37 °C, take 100 μL of the reaction solution at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h and 12 h respectively, add 100 μL of 0.2 M sodium carbonate solution to mix and terminate the reaction, and take 10 μL of the supernatant for UPLC analysis after centrifugation. UPLC is used to determine the degradation degree of the peptide.

[0072] 4.2 Thermal Stability

[0073] Dissolve antibacterial peptides VG7 and C4-VG7 in ultrapure water respectively to make the final concentration 0.1 mM, incubate at 80 °C, take 100 μL of the reaction solution at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h and 12 h respectively, quickly place on ice to cool and terminate the reaction, and conduct UPLC analysis after half an hour. UPLC is used to determine the degradation degree of the peptide.

[0074] 4.3 Stability under Different pH Conditions

[0075] Use a mixture of 0.1 M citric acid and 0.1 M sodium citrate to prepare buffers with pH 3 and pH 5, use Tris-HCl to prepare buffers with pH 7 and pH 9, and use a mixture of 0.1 M sodium bicarbonate and 0.1 M sodium carbonate to prepare buffers with pH 9.5, pH 10, pH 10.5 and pH 11. Dissolve antibacterial peptides VG7 and C4-VG7 in the prepared solutions respectively to make the final concentration 0.1 mM, incubate at 26 °C, take 100 μL of the reaction solution at 0 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h and 12 h respectively, quickly place on ice to cool and terminate the reaction, and conduct UPLC analysis after half an hour. UPLC is used to determine the degradation degree of the peptide.

[0076] The results are asFigure 2 As shown, C4-VG7 is relatively stable in simulated gastric juice and at 80 °C, highly stable at pH ≤ 9, and its stability drops rapidly when the pH is higher than 9. This indicates that the antimicrobial peptide C4-VG7 has a wide range of applications and can be used in various environments.

[0077] Example 2

[0078] Antibacterial Activity of Antimicrobial Peptides and Antimicrobial Peptides Modified with Fatty Chains

[0079] 1. Determination of MIC of Antimicrobial Peptides and Common Antibiotics

[0080] The minimum inhibitory concentration (MIC) is the minimum drug concentration that inhibits the growth of 99.9% of bacteria and is an important indicator for evaluating the antibacterial activity of drugs. The specific steps for MIC determination are as follows:

[0081] Prepare MH broth medium, LB broth medium, and LB solid medium according to the formula, sterilize at 121 °C under high-pressure steam for 15 min, and place in the refrigerator at 4 °C for later use after cooling.

[0082] Prepare the concentrations of the antimicrobial peptides to be tested and positive drugs (streptomycin, kanamycin, polymyxin B, vancomycin, and cefoperazone sodium) to be 256 μM, and place in the refrigerator at 4 °C for later use.

[0083] Take the strains stored at -80 °C, streak inoculate them on LB solid medium, and culture at 37 °C for 20 h. After the bacteria grow well, seal them with a sealing film and place them upside down in the refrigerator at 4 °C for later use. During the experiment, pick a single colony into 30 mL of MH broth medium and culture overnight at 37 °C and 200 rpm until the bacteria are in the logarithmic growth phase. After the bacteria grow to the logarithmic growth phase, adjust the number of bacteria to 1×10 6 CFU / mL using MH broth medium, and determine the minimum inhibitory concentration of the drug using the two-fold dilution method. Take a 96-well plate, add 100 μL of MH broth medium to the 12th column as a blank control (use LB broth medium as a blank control when measuring fungi), and add 100 μL of diluted bacterial solution to wells 1-11 in rows A-H. After inoculating the bacterial solution, add 20 μL of the antimicrobial peptide to be tested to wells 1-6 in row A, with 3 replicates for each drug, and add 20 μL of the positive drug to wells 7-11 as a positive control (since the resistance of clinical multidrug-resistant Acinetobacter baumannii is known, only the MIC value of the antimicrobial peptide against clinical multidrug-resistant Acinetobacter baumannii is tested here). After mixing, aspirate 100 μL and add it to the corresponding wells in row B, mix, and so on. Finally, aspirate 100 μL of the bacterial solution from row H and discard it. The final drug concentrations are successively halved from 1 to 128 μM. Incubate continuously in a constant temperature incubator at 37 °C for 20 h.

[0084] Measure the absorbance at 600 nm (OD600 )。The blank control wells remained clear throughout the experiment, indicating that the entire experiment was not contaminated by bacteria; the test wells were turbid, indicating that bacterial growth was not inhibited. The lowest drug concentration corresponding to the clear test wells was the MIC of the tested drug.

[0085] The results are as Figure 3 shown. VG7 has excellent inhibitory effects on both Gram-positive and Gram-negative bacteria. On this basis, VG7 was modified with fatty acid chains to obtain the analogue C4-VG7. After testing, the MIC values of C4-VG7 against various bacteria were all not more than 1 μM, showing more excellent broad-spectrum antibacterial activity.

[0086] 2. Time-kill curves of antimicrobial peptides VG7 and C4-VG7 against four bacteria

[0087] After the bacteria grew to the logarithmic growth phase, the number of bacteria was adjusted to 1×10 6 CFU / mL using MH broth medium. 100 μL of the bacterial solution and 100 μL of the antimicrobial peptide or fatty acid chain-modified antimicrobial peptide to be tested (concentration: 8×MIC) were added to row A of a 96-well plate. Each drug was repeated in 3 wells and mixed well by pipetting. 160 μL of MH broth medium was added to rows B - D. At 0 min, 2 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min, 90 min, 120 min, 150 min, and 180 min, 40 μL of the solution was taken from row A and added to the wells in row B, mixed well by pipetting, then 40 μL of the solution was taken and added to row C, and serially diluted five-fold to row D in sequence. 50 μL of the bacterial solution at each dilution was inoculated onto a Petri dish with LB solid medium and incubated upside down in an incubator at 37 °C for 20 h. Colony counting was performed to estimate the concentration of viable bacteria after the bacteria were treated with each drug for different times, the corresponding log10 values were calculated, and the bactericidal kinetic curves were plotted. The above experimental operations were repeated three times.

[0088] The results are as Figure 4 shown. Both VG7 and C4-VG7 could kill the four bacteria within 3 hours. In particular, Acinetobacter baumannii and Pseudomonas aeruginosa were completely killed within 10 minutes and 30 minutes respectively, demonstrating the rapid bactericidal ability of the antimicrobial peptides.

[0089] 3. Inhibition and eradication of biofilm formation of antimicrobial peptides VG7 and C4-VG7 against four bacteria

[0090] A biofilm is a structured colony that attaches to the surface of an instrument and is encapsulated in an extracellular matrix (ECM). Compared with planktonic bacteria that are not attached to a surface, the bacteria in a biofilm exhibit stronger antibiotic tolerance.

[0091] The specific steps are as follows:

[0092] 1) Inhibiting the formation of bacterial biofilms: After the bacteria grow to the logarithmic growth phase, use MH broth medium to adjust the number of bacteria to 1×10 6 CFU / mL, add them into 96-well plates, 100 μL per well, then add an equal volume of antibacterial peptide solutions or fatty acid chain-modified antibacterial peptide solutions with different concentrations and set up negative controls. Set 3 replicates for each group and place them in a 37°C constant temperature incubator for 24 h. After the incubation, discard the culture medium and gently wash each well 3 times with PBS buffer. Then add 100 μL of 0.1% crystal violet solution to each well and stain at room temperature for 15 min. Discard the staining solution and gently wash each well 3 times with PBS buffer. Add 200 μL of 33% glacial acetic acid solution to each well to dissolve the stained biofilm. Use a microplate reader - microplate detection system Spectra M2 to measure the absorbance value of each well at a wavelength of 570 nm. Compare the absorbance values of each group to evaluate the inhibitory effect of antibacterial peptides and fatty acid chain-modified antibacterial peptides on biofilm formation.

[0093] 2) Removing the pre-formed bacterial biofilms: After the bacteria grow to the logarithmic growth phase, use MH broth medium to adjust the number of bacteria to 1×10 6 CFU / mL, add them into 96-well plates, 100 μL per well, and place them in a 37°C constant temperature incubator for 24 hours to form biofilms. After the incubation, discard the culture medium and gently wash each well 3 times with PBS buffer. Then add 100 μL of antibacterial peptide solutions or fatty acid chain-modified antibacterial peptide solutions with different concentrations to each well, and set up a negative control group without adding antibacterial peptides or fatty acid chain-modified antibacterial peptides. Set 3 replicates for each group and place them in a 37°C constant temperature incubator for 24 h. After the incubation, discard the culture medium and gently wash each well 3 times with PBS buffer. Then add 100 μL of 0.1% crystal violet solution to each well and stain at room temperature for 15 min. Discard the staining solution and gently wash each well 3 times with PBS buffer. Add 200 μL of 33% glacial acetic acid solution to each well to dissolve the stained biofilm. Use a microplate reader - microplate detection system Spectra M2 to measure the absorbance value of each well at a wavelength of 570 nm. Compare the absorbance values of each group to evaluate the removal effect of antibacterial peptides and fatty acid chain-modified antibacterial peptides on the pre-formed biofilms.

[0094] As Figure 5 shown, both VG7 and C4-VG7 can significantly inhibit the formation of bacterial biofilms in a concentration-dependent manner. As Figure 6 shown, both VG7 and C4-VG7 can significantly remove the pre-formed biofilms of bacteria other than Acinetobacter baumannii.

[0095] 5. Evaluation of the drug resistance of antibacterial peptides VG7 and C4-VG7 against four bacteria

[0096] Antimicrobial peptides have lower drug resistance compared to traditional small molecule drugs. In this invention, MRSA was continuously cultured at sub-MIC concentrations to evaluate whether antimicrobial peptides are prone to inducing drug resistance in MRSA.

[0097] After the bacteria grew to the logarithmic growth phase, they were diluted to 2×10 6 CFU / mL, added to a 96-well plate, and the drug was added using the two-fold dilution method to make the final drug concentration 0.25 - 4×MIC. Three replicates were set for each drug. Continuous culture was carried out at 37°C for 12 h and the MIC was recorded. The growth of the bacteria was observed. The bacterial solution with the second highest antibiotic concentration (showing visible growth) in the culture solution was diluted 1000-fold in MH broth medium, added to a 96-well plate, and the drug was added using the two-fold dilution method. The drug concentration was determined according to the MIC of the drug against the previous generation of bacteria. The above operations were repeated for 42 passages, and the changes in MIC were recorded.

[0098] The results are as Figure 7 shown. Compared with traditional antibiotics, antimicrobial peptides VG7 and C4-VG7, especially C4-VG7, are less likely to induce drug resistance in bacteria.

[0099] 6. Hemolytic toxicity determination

[0100] Blood from mice was collected using a disposable vacuum blood collection tube containing EDTA and thoroughly mixed. Centrifugation was carried out at 1200 rpm for 10 min at 4°C. The lower layer of erythrocytes after centrifugation was washed twice with PBS and resuspended in PBS to prepare a solution containing 5% blood cells, which was plated in a 96-well plate, 100 μL per well. Equal volumes of prepared antimicrobial peptides and fatty acid chain-modified antimicrobial peptides with different concentrations were respectively added to the 96-well plate containing 5% mouse erythrocytes. Three replicates were set for each group of drugs. At the same time, an equal volume of PBS was added as a negative control, and an equal volume of Triton X-100 with a final concentration of 0.1% was added as a positive control. Incubation was carried out in a 37°C incubator for 1 h. Centrifugation was carried out at 1200 rpm for 10 min at 4°C, and the supernatant was aspirated and transferred to another 96-well plate. The absorbance at 576 nm was measured using an enzyme-linked immunosorbent assay - microplate detection system Spectra M2. The hemolytic toxicity was calculated according to the following formula:

[0101]

[0102] The results are as Figure 8 shown. The hemolytic toxicity of the fatty acid chain-modified C4-VG7 is slightly higher than that of VG7, but even at the highest concentration of 128 μM, the hemolytic toxicity of C4-VG7 is less than 5%, indicating that the potential hemolytic toxicity of the antimicrobial peptides of this invention is negligible at effective antibacterial concentrations.

[0103] 7. Scanning electron microscopy observation of the effects of antimicrobial peptides VG7 and C4-VG7 on the morphology of four bacteria

[0104] After the bacteria grew to the logarithmic growth phase, they were centrifuged (3000g, 10 min) with PBS buffer for washing three times and then resuspended in PBS buffer to a concentration of 1×10 8 CFU / mL. The bacteria and the antimicrobial peptide (PBS was added in the blank control) were co-incubated in a 37°C incubator for 2 h. After incubation, they were centrifuged (5000g, 10 min) with PBS for washing twice to remove the residual drug and then resuspended in PBS. 10 μL was taken and placed on a silicon wafer, and fixed overnight with Gluta electron microscopy fixative. The fixative was removed, and the bacteria were dehydrated stepwise with 50%, 70%, 90%, and 100% ethanol solutions, with each dehydration step lasting 10 min, and then dehydrated with 100% ethanol for 15 min. Subsequently, they were replaced with an ethanol / tert-butanol (1:1, v / v) mixed solution for 15 min, and finally replaced with tert-butanol for 10 min. They were dried at 37°C for 60 min. The samples were sputter-coated with gold using an ion sputter coater, and the morphology of the samples was observed using an environmental scanning electron microscope.

[0105] The results were as Figure 9 shown. After treatment with the antimicrobial peptide C4-VG7, a large number of MRSA cells significantly lost cell integrity, and different degrees of membrane damage were shown for Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa, with the cell membrane shrinking or breaking.

[0106] 8. Effects of antimicrobial peptide C4-VG7 on the outer membrane permeability of three kinds of bacteria

[0107] After the bacteria grew to the logarithmic growth phase, they were centrifuged at 1000g for 5 min using HEPES buffer (5 mM HEPES, 5 mM glucose, pH 7.4), washed three times and then resuspended in HEPES buffer to make the OD 600 = 0.5. NPN was dissolved in DMSO and diluted to a working solution concentration of 20 μM with PBS, and added to the bacterial solution to a final concentration of 10 μM. After dark incubation for 5 min, it was added to a 96-well black plate, and then an equal volume of an antimicrobial peptide solution with a concentration of 8 μM was added, and a PBS negative control was set. Each group had 3 replicates. The fluorescence intensity of NPN was measured within 16 min using an enzyme-linked immunosorbent assay - microplate detection system Spectra M2, with an excitation wavelength λ = 350 nm and an emission wavelength λ = 420 nm.

[0108] The results were as Figure 10 shown. After treatment with the fatty chain modifier C4-VG7, the fluorescence intensity of NPN increased significantly, showing the same trend as the positive group and the polymyxin B treatment group, indicating that C4-VG7 changed the permeability of the bacterial outer membrane.

[0109] 9. Effects of antimicrobial peptide C4-VG7 on the inner membrane permeability of four kinds of bacteria

[0110] After the bacteria grew to the logarithmic growth phase, they were centrifuged at 1000 g for 5 min using HEPES buffer (5 mM HEPES, 20 mM glucose, pH 7.4), washed three times and resuspended in HEPES buffer to make OD 600 = 0.5. 90 μL of the bacterial solution was added to each well of a 96-well plate, then an equal volume of an antibacterial peptide solution with a concentration of 8 μM was added and a PBS negative control was set. Then, 20 μL of PI dye was added to each well. The PI fluorescence intensity was measured within 45 min at 37 °C using a microplate detection system SpectraM2 of a microplate reader, with an excitation wavelength λ = 535 nm and an emission wavelength λ = 617 nm.

[0111] The results were as Figure 11 shown. After treatment with the fatty acid chain modifier C4-VG7, the PI fluorescence intensity in MRSA increased slightly, indicating that C4-VG7 did not strongly change the inner membrane permeability of MRSA; the PI fluorescence intensity in Klebsiella pneumoniae hardly changed, indicating that C4-VG7 did not change its inner membrane permeability; in Acinetobacter baumannii and Pseudomonas aeruginosa, C4-VG7 showed the same trend as the positive group and the polymyxin B treatment group, and the PI fluorescence intensity increased significantly within a short time, indicating that C4-VG7 could rapidly change the inner membrane permeability of these two bacteria.

[0112] 10. Effect of antibacterial peptide C4-VG7 on the cell membrane potential of four bacteria

[0113] After the bacteria grew to the logarithmic growth phase, they were centrifuged at 1000 g for 5 min using HEPES buffer (5 mM HEPES, 20 mM glucose, 100 mM KCl, pH 7.4), washed three times and resuspended in HEPES buffer to make OD 600 = 0.3. DiSC3(5) was dissolved in DMSO and diluted to the working solution concentration with PBS and added to the bacterial solution to make its final concentration 2 - 5 μM. The cell and dye mixture was incubated in the dark for about 30 min to ensure that the dye entered the cell membrane. Then, an equal volume of an antibacterial peptide solution with a concentration of 8 μM was added and a PBS negative control was set. Three replicates were set for each group. The fluorescence intensity of DiSC3(5) was measured every 1.5 min within 30 min using a microplate detection system SpectraM2 of a microplate reader, with an excitation wavelength λ = 622 nm and an emission wavelength λ = 673 nm. The above experimental operations were repeated three times.

[0114] The results were as Figure 12As shown in the figure, after treatment with the fatty acid chain modifier C4-VG7, the fluorescence intensity of DiSC3(5) in MRSA, Acinetobacter baumannii, and Escherichia coli increased significantly, indicating that C4-VG7 rapidly affects the membrane depolarization process of these three bacteria in a short time, thereby affecting their normal physiological activities. However, no change in the fluorescence intensity of DiSC3(5) in Pseudomonas aeruginosa was observed, indicating that C4-VG7 has no effect on its membrane potential.

[0115] 11. Antibacterial peptide C4-VG7 can bind to the DNA of four bacteria

[0116] After the bacteria grew to the logarithmic growth phase, the genomic DNA of MRSA, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa was extracted using a bacterial genomic DNA extraction kit (Vazyme, FastPure Bacteria DNA Isolation Mini Kit) and diluted to 40 μg / mL. Then, an equal volume of the polypeptide solution was added, and the final concentrations were 0.5 - 8×MIC, respectively. The mixture was incubated at 37 °C for 30 minutes. Subsequently, 9 μL of the mixture was taken and 1 μL of 10× loading buffer (Takara) was added. Then, electrophoresis was performed at a constant voltage of 90 V for 30 minutes on a 1% agarose gel in 1× TAE buffer. To ensure accuracy, 5 μL of DL10,000 DNA Marker (Takara, Japan) was added as a control. The gel image was observed and analyzed using a FluorChem E chemiluminescence gel imager (ProteinSimple).

[0117] The results are as Figure 13 shown. Compared with the blank group, when the concentration of C4-VG7 increased, the DNA bands gradually darkened and remained in the gel wells, indicating that the antibacterial peptide C4-VG7 can bind to bacterial DNA, thereby inhibiting the normal physiological activities of bacteria.

[0118] Example 3

[0119] In vivo experiment

[0120] 1. Efficacy of the acute peritoneal inflammation model induced by Acinetobacter baumannii

[0121] Six to eight-week-old SPF-grade female ICR mice after one week of adaptive feeding were induced to have neutropenia by intraperitoneal injection (i.p.) of 150 mg / kg cyclophosphamide on the 1st and 4th days. On the 5th day, the mice were inoculated by intraperitoneal injection (i.p.) with 1×10 8CFU bacteria, and then the mice were randomly divided into groups of 8. Within 1 h after injection, the mice were respectively given an equal amount of normal saline (negative control), antimicrobial peptide (0.5 mg / kg and 1 mg / kg), or positive drug (positive control, polymyxin B, 1 mg / kg). The status of the mice was observed every 12 hours. After 48 hours, the mice were euthanized and dissected, and the liver, spleen, lung, and kidney were collected respectively. Half of the collected organs were carefully placed in 1 mL of pre-cooled sterile normal saline and placed on ice, and then homogenized with a homogenizer. After appropriate dilution, 50 μL of the diluted homogenate was pipetted onto a culture dish containing LB solid medium, and it was inverted and continuously cultured in a 37 °C biochemical incubator for 24 h, and colony counting was performed and log10(CFU / g) in the corresponding tissues was calculated.

[0122] The results are as Figure 14 shown. The change in body weight of the mice in the antimicrobial peptide C4-VG7 treatment group was comparable to that in the normal saline treatment group and the positive drug polymyxin B treatment group, and the body weight showed a slight decrease. Compared with the 50% survival rate of the normal saline treatment group, the survival rate of the mice in the antimicrobial peptide C4-VG7 treatment group and the positive drug polymyxin B treatment group was 100%. Compared with the normal saline treatment group, the C4-VG7 administration group had the same therapeutic effect as the positive drug polymyxin B treatment group, and significantly reduced the bacterial load in the internal organs of the mice. These results indicate that the antimicrobial peptide C4-VG7 can exert an antibacterial effect in vivo and treat acute peritoneal inflammation caused by bacteria.

[0123] 2. Efficacy of the Acinetobacter baumannii skin infection mouse model

[0124] Six- to eight-week-old SPF-grade female ICR mice after one week of adaptive feeding were randomly divided into groups of 9. They were anesthetized by intraperitoneal injection of 75 mg / kg of sodium pentobarbital, and the back hair was shaved off. A 1×1 cm 2 skin injury was made on the back of the mice with a sterile needle, and then 10 μL of a bacterial suspension with a concentration of 5×10 8 CFU / mL was dropped on the wound. Two hours after infection, 10 μL of normal saline (negative control), antimicrobial peptide (5 mg / mL and 10 mg / mL), or positive drug (positive control, polymyxin B, 10 mg / mL) was applied to the infected skin. After 48 hours, the mice were euthanized, and skin samples were taken and placed in 1 mL of pre-cooled sterile normal saline and placed on ice, and then homogenized with a homogenizer. After appropriate dilution, 50 μL of the diluted homogenate was pipetted onto a culture dish containing LB solid medium, and it was inverted and continuously cultured in a 37 °C biochemical incubator for 24 h, and colony counting was performed and log10(CFU / g) in the corresponding tissues was calculated.

[0125] The results are as Figure 15As shown, the change in body weight of mice in the antimicrobial peptide C4-VG7 treatment group was comparable to that in the normal saline treatment group and the positive drug polymyxin B treatment group, and the body weight remained basically unchanged. Compared with the normal saline treatment group, the C4-VG7 administration group had the same therapeutic effect as the positive drug polymyxin B treatment group, significantly reducing the bacterial load on the skin of mice. These results indicate that the antimicrobial peptide C4-VG7 can exert antibacterial effects on the body surface and treat skin infections caused by bacteria.

[0126] 3. In vivo safety assessment

[0127] Six- to eight-week-old SPF-grade female ICR mice after one week of adaptive feeding were randomly divided into groups of 6 mice each. After weighing and recording, the mice were respectively given an equal amount of normal saline (negative control), antimicrobial peptide (0.5 mg / kg and 1 mg / kg), or positive drug (positive control, polymyxin B, 1 mg / kg). The drugs were administered continuously for 5 days. The mice were weighed before each drug administration, and the changes in body weight were recorded. The activity of the mice was observed for 30 min after drug administration. Twenty-four hours after the last drug administration, the mice were weighed, blood was collected, and they were immediately sacrificed. The blood samples were centrifuged at 1200 rpm for 10 min, and the supernatant was taken. Subsequently, alanine aminotransferase (ALT / GPT), aspartate aminotransferase (AST / GOT), blood urea nitrogen (BUN), albumin (ALB), creatinine (CREA), and uric acid (UA) in the serum were detected according to the instructions of the kit.

[0128] After sacrificing the mice treated with the above-mentioned drug, the heart, liver, lungs, spleen and kidneys were immediately collected. After thoroughly washing the collected organs with pre-cooled sterile normal saline, the organs were completely immersed in 4% paraformaldehyde for at least 48 h. Subsequently, gradient dehydration of the collected organs was carried out using ethanol of different concentrations. The immersion process was as follows: dehydration with 75% ethanol for 4 h, dehydration with 85% ethanol for 2 h, dehydration with 95% ethanol for 1 h, dehydration with 100% ethanol for 0.5 h, replacing with fresh 100% ethanol, and repeating the 100% ethanol dehydration process three times. After dehydration, the samples were immersed in xylene to remove ethanol from the tissues, and this process was repeated once. After permeabilization, the samples were placed in two wax boxes for sequential wax infiltration and air drying. Subsequently, sections were cut on a fully automatic paraffin slicer, spread in a constant temperature water bath, picked up on slides, baked, and then subjected to HE staining. The samples were dewaxed, and the process was the opposite of the dehydration process until hydration with primary water. Subsequently, the slides were immersed in hematoxylin staining solution for 10 - 20 min, rinsed under tap water for 3 min, and the staining condition was observed under a microscope to see if it was appropriate. When the staining condition was appropriate, differentiation was carried out with 1% hydrochloric acid ethanol solution for 5 - 10 s, and then the slides were placed in a constant temperature water bath until the slides turned blue. The staining of the cell nucleus by hematoxylin was completed. Subsequently, the cytoplasm was stained with eosin staining solution. The slides were placed in eosin staining solution for 3 - 5 min, rinsed under tap water for 3 min, and the staining condition was observed under a microscope. Finally, gradient ethanol dehydration was carried out again, dehydration with 95% ethanol for 5 min, repeated once; dehydration with absolute ethanol for 5 min, repeated once; permeabilization with xylene for 5 min, repeated once; air dry the slides. After adding neutral balsam for sealing, the slides were sealed. Image acquisition was carried out under a digital slide scanner, and images magnified 40 times were acquired.

[0129] The results are as Figure 16 shown. The change in body weight of the mice in the antimicrobial peptide C4-VG7 administration group was comparable to that in the normal saline administration group and the positive drug polymyxin B administration group, with a slight increase in body weight. Compared with the normal saline treatment group, there were no significant changes in alanine aminotransferase (ALT / GPT), aspartate aminotransferase (AST / GOT), blood urea nitrogen (BUN), albumin (ALB) and creatinine (CREA) in the serum of the C4-VG7 administration group and the positive drug polymyxin B administration group. However, there were significant differences in the content of uric acid (UA) among the groups. The results of HE staining of visceral sections showed that the administration of antimicrobial peptide C4-VG7 caused mild inflammation in the viscera of mice, while the administration of polymyxin B led to severe inflammation in the viscera of mice, even inducing necrosis.

[0130] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and purposes of the present invention.

Claims

1. An antibacterial peptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. A method for synthesizing the antibacterial peptide according to claim 1, characterized in that, It includes the following steps: After removing the Fmoc protecting group from the α-helical peptide chain in conotoxin GeXIVA, amino acids are successively coupled in the order shown in SEQ ID NO.1 and iterated until the extension of the target peptide chain is completed, and then the protecting groups are removed to obtain the antibacterial peptide.

3. A fatty acid chain-modified antimicrobial peptide, characterized in that, It is obtained by coupling the exposed amino group of the antibacterial peptide described in claim 1 with a fatty acid. The amino acid sequence of the fatty chain-modified antibacterial peptide is shown in SEQ ID NO.

2.

4. The fatty acid chain-modified antimicrobial peptide according to claim 3, wherein After the coupling reaction is completed, the coupling reaction product is separated by the ether precipitation method to obtain a crude peptide.

5. The fatty acid chain-modified antibacterial peptide according to claim 3, characterized in that, The synthesis of the fatty chain-modified antibacterial peptide includes purification, and the specific operation steps are as follows: Trifluoroacetic acid and water are mixed at a volume ratio of 0.1:100 as mobile phase A, and acetonitrile and water are mixed at a volume ratio of 3:2 as mobile phase B. Gradient elution separation is carried out by high performance liquid chromatography. During the gradient elution process, mobile phase B linearly changes from 5% to 70% in 0 - 65 minutes; Trifluoroacetic acid and water are mixed at a volume ratio of 0.1:100 as mobile phase A, and acetonitrile and water are mixed at a volume ratio of 9:1 as mobile phase B. Reverse phase chromatography is used to detect the purity of the separated compound. Mobile phase B linearly changes from 5% to 70% in 0 - 30 minutes; Compounds with a purity > 95% are collected and dried to obtain the fatty chain-modified antibacterial peptide.

6. Use of the antibacterial peptide described in claim 1 or the fatty chain-modified antibacterial peptide described in claim 3 in the preparation of antibacterial products.

7. The use according to claim 6, characterized in that, The antibacterial effect is against Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

8. An antibacterial product, characterized in that, It uses the antibacterial peptide described in claim 1 or the fatty chain-modified antibacterial peptide described in claim 3 as the sole active ingredient.

9. The antibacterial product according to claim 8, wherein The antibacterial product uses the combination of the antibacterial peptide described in claim 1 and the fatty chain-modified antibacterial peptide described in claim 3 as the sole active ingredient.

10. The antibacterial product according to claim 8 or 9, characterized in that, The antibacterial product includes pharmaceutically acceptable excipients.

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